Reproductive efficiency, which can be defined as the number of pigs produced per breeding female, is the major limiting factor in the efficient production of pork. The number of pigs born alive in the United States averages approximately 9.5 pigs per litter. Heritability for litter size is low (10% -15%), and standard genetic methods of selecting breeding females on the basis of past litter size have not been effective. Therefore, there is a need for an approach that deals with selection for reproduction at the cellular or DNA level.
Chinese breeds are known for reaching puberty at an early age and for their large litter size. American breeds are known for their greater growth rates and leanness. Thus, it would be desirable to combine the best characteristics of both types of breeds, thereby improving the efficiency of U.S. pork production. These efforts would be greatly assisted by the discovery of genes or genetic markers that are associated with increased litter size in pigs.
Reproduction in mammals takes place in response to a chain of events that occur between the brain and the reproductive organs. The steroid hormones, such as estrogen, play a crucial role. Steroid hormones interact with cells and tissues, initiating a series of events that result in the ability to reproduce successfully.
In pigs, estrogen, which is produced mainly by the ovaries, has profound effects on the uterus, brain, and pituitary gland. Estrogens modulate the onset of puberty, reproductive behaviors, cyclic release of gonadotropins, and feeding behavior. The effects of estrogens take place as a result of the binding of estrogen to specific receptor proteins found in the nucleus of the estrogen-responsive cells. McEwen, et al., Recent Prog. Horm. Res., 38:41-92 (1982), incorporated herein by reference.
The gene responsible for coding for the human estrogen receptor has been identified, and it is publicly available from the American Type Culture Collection. See ATCC Catalog Sept. 1990, page 112, entry 57681, incorporated herein by reference. The probe is named pOR3 and is 1.3 kb. Green et al., Nature (London) 320:134-139 (1986), incorporated herein by reference. The human gene is known to be polymorphic as a result of restriction fragment length polymorphism (RFLP) analysis. Castagnoli et al., Nucl. Acids Res., 15:886 (1987) and Coleman et al., Nucl. Acids Res., 16:7208 (1988), both of which are incorporated herein by reference. The functional differences relating to these different genotypes are not well understood, but they have been implicated in increased spontaneous abortions in humans with breast cancer. Lehrer et al., The Lancet, 335:622-624 (Mar. 17, 1990), incorporated herein by reference.
The estrogen receptor gene has been isolated and sequenced for other species, but not for pigs. Koike et al., Nucl. Acids Res., 15:2499-2513 (1987), incorporated herein by reference, reports the isolation and sequencing of a cDNA clone of the rat uterus estrogen receptor. The authors state that a comparison of rat, human, and chicken estrogen receptor sequences indicates the presence of three highly conserved regions, suggesting that these regions play important roles in estrogen receptor function.
In addition, Koike et al. Biochemistry 26:2563-2568 (1987), incorporated herein by reference, reports the partial characterization of the porcine estrogen receptor binding site. The paper reports a fragment of about 30 kDa that probably corresponds to the hydrophobic C-terminal-half region and has a greater than 90% homology with the corresponding rat, human, and chicken sequences.
RFLP analysis has been used by several groups to study pig DNA. Jung et al., Theor. Appl. Genet., 77:271-274 (1989), incorporated herein by reference, discloses the use of RFLP techniques to show genetic variability between two pig breeds. Polymorphism was demonstrated for swine leucocyte antigen (SLA) Class I genes in these breeds. Hoganson et al., Abstract for Annual Meeting of Midwestern Section of the American Society of Animal Science, Mar. 26-28, 1990, incorporated herein by reference, reports on the polymorphism of swine major histocompatibility complex (MHC) genes for Chinese pigs, also demonstrated by RFLP analysis. Jung et al. Animal Genetics, 20:79-91 (1989), incorporated herein by reference, reports on RFLP analysis of SLA Class I genes in certain boars. The authors state that the results suggest that there may be an association between swine SLA/MHC Class I genes and production and performance traits. They further state that the use of SLA Class I restriction fragments, as genetic markers, may have potential in the future for improving pig growth performance.
Prior to the present invention, RFLP analysis has not been applied to the pig estrogen receptor gene, which has not even been isolated or characterized. The present invention overcomes these deficiencies. It provides genetic markers, based upon the discovery of polymorphisms in the pig estrogen receptor gene, which relate to increased litter size in pigs. This will permit genetic typing of pigs for their estrogen receptor genes and determining the relationship of specific RFLPs to increased litter size. It will also permit the identification of individual males and females that would be expected to produce a litter size larger than the average for their breed. Thus, the markers will be selection tools in breeding programs to develop lines and breeds that produce litters containing a larger number of offspring.